Central to the hypoxia response is the activity of the hypoxia inducible factor alpha (HIF1alpha) which regulates a large subset of genes implicated in metabolism, cell growth, motility and cell death. HIF1alpha is exclusively functional under hypoxia conditions, since its availability is limited to these conditions, due to its tightly controlled degradation under normoxia, but not hypoxia conditions. Our studies reveal that certain tumor derived pVHL mutants retain their ability to associate with HIF-1alpha and to direct its ubiquitination, suggesting a possibility that the pVHL tumor suppressor may play a previously unidentified role in facilitating the recognition of the ubiquitinated HIF-1alpha by the 26S proteasome for degradation. Accordingly, we plan to (1) further characterize the role of the pVHL E3 in ubiquitination and degradation of HIF1alpha and advance our understanding of how this process is regulated under hypoxia. HIF1alpha degradation requires its hydroxylation on specific proline residues, a prerequisite for the association with the E3 ligase pVHL, resulting in its efficient ubiquitination followed by its proteasome-dependent degradation. In our preliminary results we obtained data to support the regulation of prolyl hydroxylases (PHD1/3) stability, by the RING finger E3 ligase protein Siah2. Siah2-targeting of PHD1/3 ubiquitination and degradation is enhanced under hypoxia conditions, thereby limiting the hydroxylation of HIF1alphaon prolines and rendering HIF1alpha stable. These findings provide the foundation to our hypothesis that as an important regulator of PHD1/3, Siah2 serves as important regulator of HIF1alpha and possibly of other proteins that are subject to regulation by PHDs. Accordingly, we plan to (2) elucidate the mechanism underlying regulation of Siah2 activity under hypoxia, (3) identify domains and requirements for Siah2-dependent ubiquitination/degradation of PHD1/3 (4) determine the role of Siah2 in tumorigenicity under hypoxia conditions, Overall these proposed studies will add novel insights to understanding the role of Siah2 and pVHL in the hypoxia-mediated and PHD-dependent regulation of HIF-1alpha. Given the important role of PHD and HIF1alpha in tumor progression and metastasis our studies will add important new insight to understanding the regulation of these processes.